US7456378B2 - Confocal microscope and multiphoton excitation microscope - Google Patents

Abstract

The invention provides a confocal microscope comprising a light source; a light scanning unit; an array device; a line-beam generating unit for imaging illumination light in the form of a straight line extending, on the array device, in a direction intersecting the scanning direction of the light scanning unit; an objective lens for imaging the illumination light reflected or transmitted at the array device on a specimen; a beamsplitter, between the array device and the light scanning unit, for splitting off from the illumination light detection light from the specimen; a two-dimensional image-acquisition unit for acquiring the split off detection light; and a control unit for controlling the light scanning unit and the array device, wherein the array device is disposed in an optically conjugate positional relationship with a focal plane of the objective lens, and the control unit performs control so as to synchronize the light scanning unit and the array device.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a confocal microscope and to a multiphoton excitation microscope.

This application is based on Japanese Patent Application No. 2006-145075, the content of which is incorporated herein by reference.

2. Description of Related Art

A confocal microscope according to the related art is disclosed, for example, in Japanese Unexamined Patent Application, Publication No. HEI-8-271792 and US Patent Application No. 2004/0113059.

Japanese Unexamined Patent Application, Publication No. HEI-8-271792 discloses a confocal microscope in which laser light is two-dimensionally scanned with two galvanometer mirrors, and fluorescence returning via the galvanometer mirrors is detected with a light detector such as a photomultiplier tube.

US Patent Application No. 2004/0113059 discloses a confocal microscope in which a digital mirror array device and a one-axis galvanometer mirror are provided in a common light path of illumination light and detection light, and a beam having a straight-line shape in cross-section is imaged on the digital mirror array device. Laser light is two-dimensionally scanned on the surface of a specimen based on the on/off operation of the digital mirror array device and the rocking motion of the galvanometer mirrors, and fluorescence returning via the digital mirror array device and the galvanometer mirror is detected by a one-dimensional line sensor.

However, when the laser light is two-dimensionally scanned with the galvanometer mirror, because the driving speed of the galvanometer mirror is delayed, a comparatively long time is required for acquiring one single image, which is not compatible with observing a fast response of the specimen.

Also, when the fluorescence is emitted from the specimen surface and returns via the galvanometer mirror, the focal position of the fluorescence focused on the light detector or the line sensor does not move, and therefore, when constructing an image, it is necessary to synchronize the light detector or the line sensor with the scanning position of the galvanometer mirror and/or the digital mirror array device. Therefore, it is necessary to perform complicated control of the light detector or the line sensor, and it is thus not possible to directly use, for example, a commercially available two-dimensional CCD camera.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a confocal microscope and a multiphoton excitation microscope which can acquire a high-resolution confocal image or multiphoton-excitation image and which can use a commercially available CCD camera.

A first aspect of the present invention is a confocal microscope comprising a light source; a light scanning unit configured to scan illumination light from the light source in one direction on a specimen; an array device in which are two-dimensionally arrayed a plurality of elements whose light reflecting or transmitting state can be electrically controlled; a line-beam generating unit configured to image the illumination light from the light source in the form of a line extending, on the array device, in a direction intersecting a scanning direction of the light scanning unit; an objective lens configured to image the illumination light reflected or transmitted at the array device on the specimen; a beamsplitter, between the array device and the light scanning unit, configured to split off from the illumination light detection light returning from the specimen via the objective lens and the array device; a two-dimensional image-acquisition unit configured to acquire the detection light split off by the beamsplitter; and a control unit configured to control the light scanning unit and the array device, wherein the array device is disposed in an optically conjugate positional relationship with a focal plane of the objective lens, and the control unit performs control so as to synchronize the light scanning unit and the array device.

According to the aspect described above, the illumination light emitted from the light source is scanned in one direction by the operation of the light scanning unit and is imaged onto the elements of the array device in the form of a straight line extending in a direction intersecting the scanning direction of the light scanning unit by the operation of the line-beam generating unit. The illumination light reflected or transmitted at the elements of the array device is focused by the objective lens and is imaged onto the specimen, which is disposed in an optically conjugate positional relationship with the array device.

Therefore, the specimen is irradiated with the entire beam imaged in the form of a straight line or a portion of the beam, according to the state of the elements. The detection light, such as reflection light reflected at the specimen or fluorescence generated by exciting the specimen, returns via the objective lens and the array device, is split off from the illumination light by the beamsplitter before it returns to the light scanning unit, and is detected by the two-dimensional image-acquisition unit. Returning the light via the array device allows the elements on the array device to function as a confocal pinhole, and therefore, it is possible to acquire a clear image of the specimen located at the focal plane of the objective lens.

In this case, because the detection light does not return to the light scanning unit but is instead split off from the illumination light, when the illumination light is scanned in one direction by the light scanning unit, the detection light is also scanned in one direction on the two-dimensional image-acquisition unit. Because the array device and the light scanning unit are synchronously controlled by the control unit, the illumination position on the specimen and the detection position of the detection light on the two-dimensional image-acquisition unit are in one-to-one correspondence. Therefore, it is possible to acquire two-dimensional image information without performing any special or complicated control in the two-dimensional image-acquisition unit, and therefore, it is possible to employ a commercially available two-dimensional image-acquisition unit, such as a CCD camera.

In the aspect of the invention described above, the beamsplitter may be disposed in an optically conjugate position with respect to a pupil position of the objective lens and may spatially separate the illumination light and the detection light.

With this configuration, compared to a case in which the light is separated based on wavelength, for example, using a dichroic mirror, it is possible to separate the illumination light and the detection light with a simple configuration, like a slit, independently of wavelength.

In the aspect of the invention described above, the control unit may control the array device so that elements where the straight-line-shaped beam is imaged and elements close thereto are in the same operating state.

With this configuration, by placing the plurality of elements close to the line beam in the same operating state, it is possible to increase the pinhole diameter, which allows a brighter image to be acquired.

In the aspect of the invention described above, the line-beam generating unit may be formed of a cylindrical lens, and the cylindrical lens may be disposed on the light source side of the light scanning unit.

With this configuration, it is possible to keep the illumination light incident on the cylindrical lens stationary, and therefore, only good on-axis performance of the cylindrical lens need be ensured. Therefore, the optical design can be simplified.

In the aspect of the invention described above, after the control unit activates only some of the elements corresponding to the position where the straight-line-shaped beam is imaged on the array device and scans the straight-line-shaped beam, the control unit may activate elements different from the above-mentioned activated elements and scan the straight-line-shaped beam.

With this configuration, it is possible to achieve a confocal effect also in the longitudinal direction of the line beam, which allows a high-resolution image to be acquired.

The aspect of the invention described above may further comprise a wavefront converting device, between the array device and the objective lens, configured to adjust a focal position on the specimen in an optical axis direction.

With this configuration, it is possible to adjust the focal position on the specimen in the optical axis direction using the wavefront conversion device, which allows three-dimensional image information to be acquired.

The aspect of the invention described above may further comprise a second light scanning unit configured to scan stimulus light, for irradiating the specimen, in one direction on the specimen; a second array device in which are two-dimensionally arrayed a plurality of elements whose light reflecting or transmitting state can be electrically controlled; a second line-beam generating unit configured to image, on the second array device, the stimulus light in the form of a straight line extending in a direction intersecting the scanning direction of the second light scanning unit; an optical component configured to establish a conjugate relationship between the second array device and a focal plane of the objective lens; and a second control unit configured to control the operating states of each element in the second array device, wherein by scanning the line beam with the second light scanning unit, the stimulus light is radiated at positions on the specimen that correspond to activated elements in the second array device.

With this configuration, the optical stimulus can be quickly applied to any plurality of points (individual points or areas), and it is therefore possible to observe a fast response of the specimen to the optical stimulus.

A second aspect of the present invention is a multiphoton excitation microscope comprising an ultrashort pulsed laser light source; a light scanning unit configured to scan ultrashort pulsed laser light from the ultrashort pulsed laser light source in one direction on a specimen; an array device in which are two-dimensionally arrayed a plurality of elements whose light reflecting or transmitting state can be electrically controlled; a line-beam generating unit configured to image the ultrashort pulsed laser light from the ultrashort pulsed laser light source in the form of a straight line extending, on the array device, in a direction intersecting the scanning direction of the light scanning unit; an objective lens configured to image, on the specimen, the ultrashort pulsed laser light reflected or transmitted at the array device; a beamsplitter, between the objective lens and the array device, configured to split off fluorescence generated in the specimen and collected by the objective lens; a two-dimensional image-acquisition unit configured to acquire the fluorescence split off by the beamsplitter; and a control unit configured to control the light scanning unit and the array device, wherein the control unit performs control so as to synchronize the light scanning unit and the array device.

According to the aspect described above, the ultrashort pulsed laser light emitted from the ultrashort pulsed laser light source is scanned in one direction by the operation of the light scanning unit and is imaged onto the elements of the array device in the form of a straight line extending in a direction intersecting the scanning direction of the light scanning unit by the operation of the line-beam generating unit. The ultrashort pulsed laser light reflected or transmitted by the elements of the array device is focused by the objective lens and is imaged at the focal plane of the objective lens, which is disposed in an optically conjugate positional relationship with the array device.

As a result, a multiphoton excitation effect is produced in the specimen and fluorescence is generated only in a thin region in the vicinity of the focal plane of the objective lens. The generated fluorescence is collected by the objective lens, is split off by the beamsplitter before reaching the array device, and is acquired by the two-dimensional image-acquisition unit.

Because fluorescence is produced only in a thin region along the focal plane due to the multiphoton excitation effect, it is possible to acquire a clear fluorescence image of the specimen.

In this case, because the detection light is split off from the ultrashort pulsed laser light without returning to the array device, when the ultrashort pulsed laser light is scanned in one direction by the light scanning unit, the detection light is also scanned in one direction on the two-dimensional image-acquisition unit. Because the array device and the light scanning unit are synchronously controlled by the control unit, the irradiation position of the ultrashort pulsed laser light on the specimen and the detection position of the fluorescence on the two-dimensional image-acquisition unit are in one-to-one correspondence. Therefore, it is possible to acquire a two-dimensional fluorescence image without conducting special or complicated control in the two-dimensional image-acquisition unit, and therefore, it is possible to employ a commercially available two-dimensional image-acquisition unit, such as a CCD camera.

The present invention affords an advantage in that it is possible to acquire a high-resolution confocal image or multiphoton excitation image and it is not necessary to perform complicated control of a detector, thus allowing a commercially available CCD camera to be used.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A and 1B are optical diagrams showing the principal components of a confocal microscope according to an embodiment of the present invention, whereinFIG. 1A is an elevational view andFIG. 1B is a plan view.

FIGS. 2A and 2B are diagrams for explaining the operating states of a digital mirror array device in the confocal microscope shown inFIG. 1A andFIG. 1B.FIG. 2A shows an operating state in which substantially an entire column at the left side is turned on, andFIG. 2B shows an operating state in which substantially an entire column near the center is turned on.

FIGS. 3A to 3D are diagrams for explaining operating states of the digital mirror array device in the confocal microscope shown inFIG. 1A andFIG. 1B.FIG. 3A andFIG. 3C show an operating state in which part of the column at the left side is turned on, andFIG. 3B andFIG. 3D show an operating state in which part of the row near the center is turned on.

FIG. 4A andFIG. 4B show operating states, similar toFIG. 2A andFIG. 2B, in which mirror elements surrounding the irradiation position of the laser light are also turned on.

FIG. 5A andFIG. 5B show operating states, similar toFIGS. 3A to 3D, in which mirror elements surrounding the irradiation position of the laser light are also turned on.

FIG. 6A andFIG. 6B show a first modification of the confocal microscope shown inFIG. 1A andFIG. 1B.

FIG. 7A andFIG. 7B are optical diagrams showing the principal components of a multiphoton excitation microscope according to an embodiment of the present invention, whereinFIG. 7A is an elevational view andFIG. 7B is a plan view.

FIG. 8A andFIG. 8B show a variable-light-stimulus confocal microscope, as a second modification of the confocal microscope shown inFIG. 1A andFIG. 1B, whereinFIG. 8A is an elevational view andFIG. 8B is a plan view.

FIG. 9 shows an operating state of the digital mirror array device during optical stimulation with the configuration inFIG. 8A andFIG. 8B.

FIG. 10A andFIG. 10B show, as a third modification of the confocal microscope shown inFIG. 1A andFIG. 1B, an example in which an image guide is disposed at the focal plane of an objective lens, whereinFIG. 10A is an elevational view andFIG. 10B is a plan view.

DETAILED DESCRIPTION OF THE INVENTION

Aconfocal microscope1 according to an embodiment of the present invention will be described below with reference to the drawings.

Theconfocal microscope1 according to this embodiment is a laser-scanning confocal microscope.

As shown inFIG. 1A andFIG. 1B, theconfocal microscope1 according to this embodiment includes a laser light source (light source; not shown in the drawing) for generating laser light L₁; alight scanning unit2 for one-dimensionally scanning the laser light L₁from the light source in one direction; a cylindrical lens (line-beam generating unit)3 for converting the laser light L₁scanned by thelight scanning unit2 into a beam imaged in the form of a straight line; a digital mirror array device (array device)4 in which a plurality of mirror elements (elements)4athat can be switched on and off are two-dimensionally arrayed; anobjective lens5 for focusing the laser light L₁reflected by themirror elements4ain the digital mirror array device4 (seeFIG. 2A andFIG. 2B) to image it at a specimen A; abeamsplitter6 for splitting off from the laser light L₁fluorescence F returning from the specimen A; two-dimensional CCD cameras (two-dimensional image-acquisition units)7 for imaging the split-off fluorescence F; and acontrol unit8 for controlling thelight scanning unit2 and the digitalmirror array device4. In order to simplify the drawings, inFIG. 1A andFIG. 1B, the reflective-type digitalmirror array device4 is replaced with a transmissive type.

Thelight scanning unit2 is, for example, an acousto-optic scanner. By changing the diffraction direction according to the frequency of an input ultrasonic wave based on a control signal from thecontrol unit8, thelight scanning unit2 changes the emission direction of the incident laser light L₁, thereby enabling scanning in one direction.

The digitalmirror array device4 is disposed in a conjugate positional relationship with respect to the focal plane of theobjective lens5. Therefore, with the focal plane of theobjective lens5 located inside the specimen A, when the straight-line-shaped laser light L₁is scanned in one direction by operating thelight scanning unit2, the laser light L₁is scanned in one direction on the specimen A and the digitalmirror array device4.

As shown inFIG. 2A andFIG. 2B, the digitalmirror array device4 is formed of the plurality of two-dimensionally arrayedmirror elements4a, which can be switched on and off. The laser light L₁incident on themirror elements4athat are set in the on state is directed towards the specimen A upon reflection by thosemirror elements4a. The laser light L₁which is incident on themirror elements4a that are set in the off state, upon reflection by thosemirror elements4a, is directed in a different direction in which it is not incident on the specimen A.

As indicated by the hatching inFIG. 2A andFIG. 2B, by turning on one column of themirror elements4acorresponding to the image-forming position of the straight-line-shaped laser beam L₁, it is possible to direct all of the incident laser light L₁towards the specimen A. The arrow in the drawings indicates a moving direction of the column ofmirror elements4athat are switched on (the scanning direction of the laser light L₁by the light scanning unit2).

As shown by the hatching inFIGS. 3A to 3D, by turning on some of themirror elements4ain the column ofmirror elements4acorresponding to the image-forming position of the straight-line-shaped laser light L₁, it is possible to direct some of the incident laser light L₁towards the specimen A.

Returning toFIG. 1A andFIG. 1B,relay lenses9 and10 are disposed between thecylindrical lens3 and thebeamsplitter6, and between thebeamsplitter6 and the digitalmirror array device4, respectively. Therelay lenses9 and10 are standard spherical-surface lenses, and therefore, the laser light L₁imaged in the form of a line by thecylindrical lens3 is relayed by therelay lens9 to be re-imaged at thebeamsplitter6, and is then relayed by therelay lens10 to be re-imaged on the digitalmirror array device4.

An image-forminglens11 is disposed between the digitalmirror array device4 and theobjective lens5. The image-forminglens11 images the laser light L₁reflected at the digitalmirror array device4 at apupil position12 of theobjective lens5.

Thebeamsplitter6 is disposed at an optically conjugate position with respect to thepupil position12 of theobjective lens5 and is provided, at the on-axis position thereof, with aslit6afor transmitting the laser light L₁converted to a line shape by thecylindrical lens3 and relayed by therelay lens9. Areflective surface6bis provided at the specimen A side of thebeamsplitter6 for reflecting the fluorescence F returning from the specimen A.

The fluorescence F reflected by thebeamsplitter6 is focused by a focusinglens13, is separated into each wavelength by adichroic mirror14, and is acquired by two two-dimensional CCDs7 in which image-acquisition surfaces are disposed at the respective image-forming positions.

Thecontrol unit8 synchronously controls thelight scanning unit2 and the digitalmirror array device4. As described above, the digitalmirror array device4 and the focal plane of theobjective lens5 are disposed in an optically conjugate positional relationship. The incident position of the laser light L₁on the digitalmirror array device4 is changed by operating thelight scanning unit2. Therefore, thecontrol unit8 can reflect the laser light L₁incident on the digitalmirror array device4 towards the specimen A by synchronizing the scanning of the laser light L₁by thelight scanning unit2 and the turning on of themirror elements4ain the digitalmirror array device4.

The operation of theconfocal microscope1 according to this embodiment, having such a configuration, will be described below.

To acquire a fluorescence image of the specimen A using theconfocal microscope1 according to this embodiment, the laser light L₁is emitted from the laser light source (not shown in the drawing). After passing via the acousto-optic scanner2 in the form of a substantially collimated beam, the laser light L₁emitted from the laser light source is imaged in the form of a straight line, extending in one direction, by thecylindrical lens3 and is relayed by therelay lens9, whereupon it is re-imaged in the form of a straight line extending in a direction orthogonal to the direction mentioned above.

Thebeamsplitter6 is disposed at this re-imaging position. Since thebeamsplitter6 is provided with theslit6afor transmitting the re-imaged laser light L₁, all of the laser light L₁passes through theslit6ain thebeamsplitter6, is relayed by therelay lens10, and is re-imaged in the form of a straight line extending in the same direction as the image at the image-forming position of thecylindrical lens3. The digitalmirror array device4 is disposed at this re-imaging position; therefore, by turning on themirror elements4athat match the image-forming position of the laser light L₁with thecontrol unit8, it is possible to reflect the incident laser light L₁to direct it towards the specimen A.FIG. 2A shows commencement of scanning of the line-shaped beam, andFIG. 2B shows the operation during scanning.

After being imaged at thepupil position12 of theobjective lens5 by the image-forminglens11, the laser light L₁directed towards the specimen A is focused by theobjective lens5 and is imaged at the focal plane thereof. Because the focal plane of theobjective lens5 and the digitalmirror array device4 are disposed in an optically conjugate positional relationship, the laser light L₁imaged at the focal plane also forms a straight-line-shaped image extending in the same direction as the laser light L₁imaged on the digitalmirror array device4.

In the specimen A, the fluorescence F is generated by exciting a fluorescent substance contained in the specimen A at each position irradiated by the laser light L₁. The generated fluorescence F is emitted in all directions; a portion thereof is collected by theobjective lens5, is substantially collimated, passes through thepupil position12 of theobjective lens5, and is imaged at the digitalmirror array device4 by the image-forminglens11. Because the digitalmirror array device4 and the focal plane of theobjective lens5 are disposed in an optically conjugate positional relationship, themirror elements4athat are turned on function as a confocal pinhole, and therefore, only the fluorescence F that is produced from the irradiation position of the laser light L₁on the focal plane of theobjective lens5 is reflected by themirror elements4athat are turned on.

The fluorescence F reflected by the turned onmirror elements4ain the digitalmirror array device4 is incident on thebeamsplitter6 after being converted to a substantially collimated beam by therelay lens10, and is reflected by thereflective surface6bof thebeamsplitter6. Because theslit6ais provided in thebeamsplitter6, part of the fluorescence is transmitted through theslit6a, but by forming theslit6ato be sufficiently small, it is possible to reflect most of the fluorescence F. Accordingly, the fluorescence F is split off from the laser light L₁.

After being focused by the focusinglens13 and split into each wavelength by thedichroic mirror14, the fluorescence F split off from the laser light L₁is acquired by the two-dimensional CCDs7. Because the image-acquisition surfaces of the two-dimensional CCDs7 are also in an optically conjugate positional relationship with the focal plane of theobjective lens5, a straight-line-shaped fluorescence image generated at the focal plane of theobjective lens5 is directly acquired by the two-dimensional CCDs7 as straight-line-shaped fluorescence images.

In this case, when the laser light L₁is scanned by operating thelight scanning unit2, the straight-line-shaped images on the digitalmirror array device4 and the focal, plane of theobjective lens5 are moved in a direction orthogonal to the direction of those images. On the other hand, because thebeamsplitter6 is disposed in an optically conjugate positional relationship with thepupil position12 of theobjective lens5, the image of the laser light L₁formed at thebeamsplitter6 does not move, even though thelight scanning unit2 is moving, and is always coincident with theslit6a.

Thus, because thecontrol unit8 synchronously controls the digitalmirror array device4 and thelight scanning unit2 in this embodiment, themirror elements4athat correspond to the image-forming position on the digitalmirror array device4, which moves according to the operation of thelight scanning unit2, are switched on. Therefore, the laser light L₁scanned by the operation of thelight scanning unit2 is always reflected by the digitalmirror array device4, and it is thus possible to move the laser light L₁imaged in the form of a straight line in the focal plane of theobjective lens5 in a direction orthogonal to the longitudinal direction thereof.

Thus, on the two-dimensional CCDs7, the image-forming positions of the fluorescence F returning from the specimen A are moved in directions orthogonal to the longitudinal direction of the fluorescence images by operating thelight scanning unit2. Therefore, by setting the image acquisition time of the two-dimensional CCDs7 to be sufficiently longer than the scanning time of thelight scanning unit2, it is possible to acquire a two-dimensional fluorescence image of the specimen A.

With theconfocal microscope1 according to this embodiment, it is possible to acquire a two-dimensional fluorescence image merely by scanning the laser light L₁imaged in the form of a straight line in one direction with thelight scanning unit2, which is formed of an acousto-optic scanner. Therefore, compared with a conventional confocal microscope in which two-dimensional scanning is achieved with two galvanometer mirrors, it is possible to acquire a two-dimensional fluorescence image more quickly. Accordingly, an advantage is afforded in that it is possible to observe a fast response of the specimen without missing it.

With theconfocal microscope1 according to this embodiment, because thecontrol unit8 synchronously controls thelight scanning unit2 and the digitalmirror array device4, it is not necessary to perform special or complicated control for the two-dimensional CCDs7, which affords an advantage in that it is possible to use commercially available CCDs. Accordingly, theconfocal microscope1 can be constructed at low cost.

In theconfocal microscope1 according to this embodiment, thebeamsplitter6 for splitting the laser light L₁and the fluorescence F is a component that spatially separates the light using theslit6aand thereflective surface6b. Therefore, an advantage is afforded in that it is possible to construct it more simply and at lower cost than a beamsplitter that splits the light based on wavelength, such as a dichroic mirror.

In theconfocal microscope1 according to this embodiment, substantially one column of themirror elements4aof the digitalmirror array device4 is turned on so as to reflect all of the straight-line-shaped laser light L₁imaged by thecylindrical lens3. In addition, when operating thelight scanning unit2 to scan the laser light L₁, the column ofmirror elements4athat is turned on in the digitalmirror array device4 is sequentially moved.

Instead of this, however, by turning on only some of themirror elements4awhere the straight-line-shaped laser light L₁is imaged, as shown inFIG. 3A toFIG. 3D, it is possible to irradiate the specimen A with one or more spots of light.

In this case, as shown inFIG. 3A andFIG. 3B, the shadedmirror elements4aare turned on to perform a first scan, then themirror elements4ato be turned on next are shifted one row, as shown inFIG. 3C andFIG. 3D, to perform a second scan. When this is repeated five times, scanning of one screen is completed. Accordingly, it is possible to acquire an image exhibiting a confocal effect also with respect to the longitudinal direction of the line beam.

In this embodiment, turning on only the column ofmirror elements4awhere the straight-line-shaped laser light L₁is imaged makes the digitalmirror array device4 function as a confocal pinhole. However, as shown by the hatching inFIGS. 4A to 5B, it is possible to simultaneously turn on not only themirror elements4aat the image-forming position, but also one or more columns of surroundingmirror elements4aneighboring them. By doing so, although the confocal effect is reduced, an advantage is afforded in that it is possible to acquire a bright fluorescence image whose depth of field is increased according to the brightness of the specimen.

In theconfocal microscope1 according to this embodiment, the laser light L₁scanned by thelight scanning unit2 is made incident on thecylindrical lens3. Instead of this, however, as shown inFIG. 6A andFIG. 6B, thecylindrical lens3 may be disposed at the laser light source side of thelight scanning unit2. With this configuration, the laser light L₁incident on thecylindrical lens3 does not move, and therefore only good on-axis performance of thecylindrical lens3 need be ensured. Therefore, an advantage is afforded in that it is possible to simplify the optical design.Reference numeral15 in the drawing is a relay lens.

In this embodiment, in theconfocal microscope1 formed by detecting the fluorescence F from the specimen A after traveling via the digitalmirror array device4, the digitalmirror array device4 functions as a confocal pinhole. Instead of this, it is possible to construct amultiphoton excitation microscope1′ in which an ultrashort pulsed laser light source (not shown in the drawing) that emits ultrashort pulses of laser light L_1′ is used, and as shown inFIG. 7A andFIG. 7B, the fluorescence F is split off by adichroic mirror16 disposed between the image-forminglens11 and the digitalmirror array device4, before it returns to the digitalmirror array device4, and is acquired by the two-dimensional CCD7. With this configuration, due to a multiphoton excitation effect, it is possible to acquire a clearer and brighter multiphoton-excitation fluorescence image.

As shown inFIG. 7A andFIG. 7B, a wavefront conversion device (deformable mirror)17 may be disposed between thedichroic mirror16 and theobjective lens5. With this configuration, the focal plane of theobjective lens5 can be moved in the optical axis direction, which allows a three-dimensional fluorescence image of the specimen A to be acquired.

As shown inFIG. 8A andFIG. 8B, adichroic mirror18 may be disposed in a collimated light path between the image-forminglens11 and theobjective lens5 for combining optical-stimulus laser light L₂with the observation laser light L₁. The optical-stimulus laser light L₂, like the observation laser light L₁, travels along a path via a light-scanning unit2′ formed of an acousto-optic scanner, acylindrical lens3′, a digitalmirror array device4′, and an image-forminglens11′, is reflected by thedichroic mirror18, and irradiates the specimen A.

Because the digitalmirror array device4′ is conjugate with respect to the specimen A, it is possible to radiate the laser light L₂only at positions on the specimen A corresponding to themirror elements4athat are turned on. In other words, by using thecontrol unit8 to turn on themirror elements4a′ corresponding to locations on the specimen A to be irradiated with stimulus light (any two-dimensional positions that are separated from each other, such as a plurality of points or areas; indicated by the shading inFIG. 9) and scan the line illumination using the acousto-optic scanner2′, the stimulus light L₂is guided towards the specimen A only at the positions of themirror elements4a′ indicated by the shading inFIG. 9. By doing so, it is possible to quickly apply the optical stimulus to a plurality of arbitrary locations (points or areas). Furthermore, the degree of freedom for setting the stimulus positions is increased.

As shown inFIG. 10A andFIG. 10B, the focal plane of theobjective lens5 may be aligned with one end of animage guide19, and a small objectiveoptical system20 may be disposed at the other end of theimage guide19. With this configuration, the tip of theimage guide19 can be inserted inside a specimen such as a living organism, allowing in vivo examination to be carried out.

Instead of the digital mirror array device, it is possible to use a device in which elements whose light reflection (transmission) state can be electrically controlled are two-dimensionally arrayed, for example, a liquid crystal matrix array.

Claims (8)

1. A confocal microscope comprising:

a light source;

a light scanning unit configured to scan illumination light from the light source in one direction on a specimen;

an array device in which are two-dimensionally arrayed a plurality of elements whose light reflecting or transmitting state can be electrically controlled;

a line-beam generating unit configured to image, on the array device, the illumination light from the light source in the form of a straight line extending in a direction intersecting a scanning direction of the light scanning unit;

an objective lens configured to image the illumination light reflected or transmitted at the array device on the specimen;

a beamsplitter, between the array device and the light scanning unit, configured to split off from the illumination light detection light returning from the specimen via the objective lens and the array device;

a two-dimensional image-acquisition unit configured to acquire the detection light split off by the beamsplitter; and

a control unit configured to control the light scanning unit and the array device,

wherein the array device is disposed in an optically conjugate positional relationship with a focal plane of the objective lens, and

the control unit performs control so as to synchronize the light scanning unit and the array device.

2. A confocal microscope according toclaim 1, wherein the beamsplitter is disposed in an optically conjugate position with respect to a pupil position of the objective lens and spatially separates the illumination light and the detection light.

3. A confocal microscope according toclaim 1, wherein the control unit controls the array device so that elements where the straight-line-shaped beam is imaged and elements close thereto are in the same operating state.

4. A confocal microscope according toclaim 1, wherein the line-beam generating unit is formed of a cylindrical lens, and the cylindrical lens is disposed on the light source side of the light scanning unit.

5. A confocal microscope according toclaim 1, wherein after the control unit activates only some of the elements corresponding to the position where the straight-line-shaped beam is imaged on the array device and scans the straight-line-shaped beam, the control unit activates elements different from the activated elements and scans the straight-line-shaped beam.

6. A confocal microscope according toclaim 1, further comprising a wavefront converting device, between the array device and the objective lens, configured to adjust a focal position on the specimen in an optical axis direction.

7. A confocal microscope according toclaim 1, further comprising:

a second light scanning unit configured to scan stimulus light, for irradiating the specimen, in one direction on the specimen;

a second array device in which are two-dimensionally arrayed a plurality of elements whose light reflecting or transmitting state can be electrically controlled;

a second line-beam generating unit configured to image the stimulus light, on the second array device, in the form of a straight line extending in a direction intersecting the scanning direction of the second light scanning unit;

an optical component configured to establish a conjugate relationship between the second array device and a focal plane of the objective lens; and

a second control unit configured to control the operating states of each element in the second array device,

wherein by scanning the line beam with the second light scanning unit, the stimulus light is radiated at positions on the specimen that correspond to activated elements in the second array device.

8. A multiphoton excitation microscope comprising:

an ultrashort pulsed laser light source;

a light scanning unit configured to scan ultrashort pulsed laser light from the ultrashort pulsed laser light source in one direction on a specimen;

an array device in which are two-dimensionally arrayed a plurality of elements whose light reflecting or transmitting state can be electrically controlled;

a line-beam generating unit configured to image, on the array device, the ultrashort pulsed laser light from the ultrashort pulsed laser light source in the form of a straight line extending in a direction intersecting the scanning direction of the light scanning unit;

an objective lens configured to image, on the specimen, the ultrashort pulsed laser light reflected or transmitted at the array device;

a beamsplitter, between the objective lens and the array device, configured to split off fluorescence generated in the specimen and collected by the objective lens;

a two-dimensional image-acquisition unit configured to acquire the fluorescence split off by the beamsplitter; and

a control unit configured to control the light scanning unit and the array device,

wherein the array device is disposed in an optically conjugate positional relationship with a focal plane of the objective lens, and

the control unit performs control so as to synchronize the light scanning unit and the array device.

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